Probing nanoscale membrane dynamics with label-free super-resolution microscopy Renee R. Frontiera, Department of Chemistry, University of Minnesota The objective of this research is to determine how the membrane environment around a given membrane protein affects its function. Membrane proteins are critical for cellular signaling and transport, and are the targets of a large number of currently-marketed pharmaceuticals. These proteins exist in a dynamic and heterogeneous membrane environment, with highly variable chemical composition, thickness, and fluidity. These membrane parameters are thought to have significant impact of protein function, but direct causative correlations between the molecular-level membrane environment and the effectiveness of processes like drug binding and signal transduction are largely unknown. This is primarily due to the challenges of studying these small proteins in their natural environment, as they have sizes on the 1-100 nm length scale, well below the traditional optical diffraction limit. The proposed research will utilize an original label-free super-resolution microscopy technique to investigate how the local environment around a membrane protein affects its function. The chemical composition of a living cellular membrane will be mapped without the need for exogenous labels by using a new technique which couples ideas from the Stimulated Emission Depletion (STED) microscopy and Femtosecond Stimulated Raman Spectroscopy (FSRS) techniques. The use of a label-free, super-resolution imaging technique with the ability to follow ultrafast reaction dynamics represents a fundamentally new and innovative approach to studying reactions in biological systems. The proposed investigations are designed to demonstrate the broad role of membrane protein environments on membrane protein function through an examination of several systems. In particular, investigations will focus on (i) agonist binding rates and efficiencies to G- protein coupled receptors; (ii) endocytosis of siRNA in various drug delivery vehicles; and (iii) ion channel activation and transport efficiency. The work will have significant impact on human health by providing a molecular-level picture of how dynamics heterogeneous local environments affect the function of membrane proteins, particularly during drug binding and signaling events. The results of the proposed studies will lead to a better understanding of how currently marketed pharmaceuticals function, and determine which specific molecular-scale factors are responsible for a wide range of responses and efficacies.